Method and agents for removing advanced glycosylation endproducts

ABSTRACT

The present invention relates to a method and associated agents for the inhibition and treatment of protein aging in animals by stimulating the bodies of the animals to increase their recognition and affinity for advanced glycosylation end products. Specifically, the method contemplates the administration of certain agents such as advanced glycosylation endproducts, such endproducts as are bound to the carrier, monokines that stimulate phagocytic cells to increase their activity toward advanced glycosylation endproducts, and mixtures of these materials either alone, or in conjunction with other co-stimulatory agents. Numerous diagnostic and therapeutic applications are defined, and pharmaceutical compositions are also contemplated.

This invention was made with partial assistance from grants from theNational Institutes of Heath and the Brookdale Foundation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is .Iadd.a reissue of U.S. Pat. No. 4,900,747,filed Sep. 3, 1987 as application Ser. No. 91,534; which is a.Iaddend.Continuation-In-Part of application Ser. No. 907,747, filedSept. 12, 1986, by the inventors herein now abandoned, which is, inturn, a Continuation-In-Part of application Ser. No. 798,032, filed Nov.14, 1985, by Anthony Cerami, Peter Ulrich, and Michael Brownlee, nowU.S. Pat. No. 4,758,583 which is, in turn, a Continuation-In-Part ofapplication Ser. No. 590,820, now U.S. Pat. No. 4,665,192 filed Mar. 19,1984 by Anthony Cerami alone. Priority under 35 U.S.C. §120 is claimedas to all of the above earlier filed applications and the disclosuresthereof are incorporated herein by reference.

RELATED PUBLICATIONS

The Applicants are co-authors of the following articles directed to thesubject matter of the present invention: "FUNCTION OF MACROPHAGERECEPTOR FOR NONENZYMATICALLY GLYCOSYLATED PROTEINS IS MODULATED BYINSULIN LEVELS", Vlassara, Brownlee and Cerami, DIABETES (1986), Vol. 35Supp.1, Page 13a; "ACCUMULATION OF DIABETIC RAT PERIPHERAL NERVE MYELINBY MACROPHAGES INCREASES WITH THE PRESENCE OF ADVANCED GLYCOSYLATIONENDPRODUCTS", Vlassara, H., Brownlee, M., and Cerami, A. J. EXP. MED.(1984), Vol. 160, pp. 197-207; "RECOGNITION AND UPTAKE OF HUMAN DIABETICPERIPHERAL NERVE MYELIN BY MACROPHAGES", Vlassara, H., Brownlee, M., andCerami, A. DIABETES (1985), Vol. 34, No. 6, pp. 553-557;"HIGH-AFFINITY-RECEPTOR-MEDIATED UPTAKE AND DEGRADATION OFGLUCOSE-MODIFIED PROTEINS: A POTENTIAL MECHANISM FOR THE REMOVAL OFSENESCENT MACROMOLECULES", Vlassara H., Brownlee, M., and Cerami, A.,PROC. NATL. ACAD. SCI. U.S.A. (Sept. 1985), Vol. 82, pp. 5588-5592;"NOVEL MACROPHAGE RECEPTOR FOR GLUCOSE-MODIFIED PROTEINS IS DISTINCTFROM PREVIOUSLY DESCRIBED SCAVENGER RECEPTORS", Vlassara, H., Brownlee,M., and Cerami, A. JOUR. EXP. MED. (1986) (in press) "ROLE OFNONENZYMATIC GLYCOSYLATION IN ATHEROGENESIS", Cerami, A., Vlassara, H.,and Brownlee, M., Journal of Cellular Biochemistry (1986), Vol. 30, pp.111-120. All of the foregoing publications are incorporated herein byreference.

BACKGROUND OF THE INVENTION

The present invention relates generally to the in vivo reaction ofanimal proteins with glucose, and particularly to the nonenzymaticglycosylation of proteins, and to methods for their in vivo inhibition.

The reaction between glucose and proteins has been known for some time,and in its earliest manifestation, was identified in the appearance ofbrown pigments during the cooking of food. This phenomenon wasidentified by Maillard in 1912, who observed that glucose and otherreducing sugars react with amino acids to form adducts that undergo aseries of dehydrations and rearrangements to form stable brown pigments.Maillard L. C. (1912) C. R. Acad. Sci., Vol. 154, pp. 66-68.

This phenomenon was found in recent years to have its parallel in vivo.Accordingly, the nonenzymatic reaction between glucose and the freeamino groups on proteins to form a stable amino 1-deoxy ketosyl adduct,known as the Amadori product, has been shown to occur with hemoglobin,wherein a rearrangement of the Amadori product formed at the aminoterminal of the beta chain of hemoglobin by reaction with glucose, formsthe adduct known as hemoglobin A₁ c. This initial reaction of theMaillard sequence was also found to occur with a variety of other bodyproteins, such as lens crystallins, collagen and nerve proteins. SeeBunn, H. F., Haney, D. N., Gabbay, K. H. and Gallop, P. H., (1975)Blochem. Biophys. Res. Comm., Vol. 67, pp. 103-109; Koenig, R. J.,Blobstein, S. H. and Cerami, A., (1977) J. Biol. Chem., Vol. 252, pp.2992-2997; Monnier, V. M. and Cerami, A., (1983) MAILLARD REACTION INFOOD AND NUTRITION, ed. Waller, G. A., American Chemical Society, Vol.215, pp. 431-448; and Monnier, V. M. and Cerami, A., (1982) Clinics inEndocrinology and Metabolism, Vol. 11, pp. 431-452.

Moreover, brown pigments with spectral and fluorescent propertiessimilar to those of late-stage Maillard products have also been observedin vivo in association with several long-lived proteins, such as lensproteins and collagen from aged individuals. An age-related linearincrease in pigment was observed in human dura collagen between the agesof 20 and 90 years. See Monnier, V. M. and Cerami, A., (1981) SCIENCE,Vol. 211, pp. 491-493; Monnier, V. M. and Cerami, A., (1983) BlOCHEM.BIOPHYS. ACTA., Vol. 760, pp. 97-103; and Monnier, V. M., Kohn, R. R.and Cerami, A., "Accelerated Age-related Browning of Human Collagen inDiabetes Mellitus", (1984) PROC. NAT. ACAD. SCI., Vol. 81, pp. 583-587.Interestingly, the aging of collagen can be mimicked in vitro by thecross-linking induced by glucose; and the capture of other proteins inthe formation of adducts by collagen, also noted, is theorized to occurby a cross-linking reaction, and is believed to account for the observedaccumulation of albumin and antibodies in kidney basement membrane andcholesterol-bearing low density lipoprotein in the arterial wall. See,Brownlee, M., Pongor, S. and Cerami, A., (1983) J. EXP. MED., Vol. 158,pp. 1739-1744; and Kohn, R. R., Cerami, A. and Monnier, V. M., (1984)DIABETES, Vol. 33, No. 1, pp. 57-59. Cerami, A., Vlassara, H., andBrownlee, M., (1985) METABOLISM, Vol. 34, pp. 37-44.

In parent application Ser. No. 590,820 and in Pongor, S. M. et al,Supra., both incorporated herein by reference, a fluorescent chromophorewas isolated and identified which was found to be present in certainbrowned polypeptides such as bovine serum albumin and poly-L-lysine, andwas assigned a structure 2-(2-furoyl)-4(5)-2-furanyl)-1H-imidazole(hereinafter "FFI"). The compound was found to exist in a tautomericstate and has incorporated in its structure two peptide-derived aminenitrogens. The incorporation of these amine nitrogens and two glucoseresidues in the compound suggested that its peptide-bound precursor maybe implicated in the in vivo cross-linking of proteins by glucose whichis observed in the late stage of the Maillard process. See, Chang, J. C.F., Ulrich, P. D., Bucala, R., and Cerami, A., (1985) J. Biol. Chem.,Vol. 260, pp. 7970-7974. This chromophore made possible theidentification of the advanced glycosylation endproducts and assistedadditional investigations seeking to clarify the protein aging processand if possible, to identify the specific chemistry involved to assistto develop methods and agents for its inhibition. Such method and agentswere initially investigated and have been disclosed in copendingapplication Ser. No. 798,032, the disclosure of which is incorporatedherein by reference.

Further work since the development of the inhibitors in the lastmentioned copending Application resulted in the identification of whatappears to be an endogenous means for the in vivo elimination or removalof advanced glycosylation endproducts. This has been set forth in mostrecent application Ser. No. 907,747. Further development of this conceptis now presented herein, and it is accordingly to this purpose that thepresent Application is directed.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method and associated agentsare disclosed for the inhibition and treatment of protein aging inanimals by stimulating the bodies of such animals to increase theirrecognition of and affinity for advanced glycosylation endproducts. Inparticular, phagocytic cells such as monocytes and macrophages aretreated with an agent capable of causing the phagocytic cells toincrease their activity of recognizing and removing macromolecules suchas target proteins.

The agents of the present invention comprise one or more stimulatorcompounds in turn, comprising a natural or synthetic advancedglycosylation endproduct alone or bound to a carrier, said carrierincluding a material selected from carbohydrates, proteins, syntheticpolypeptides, lipids, bio-compatible natural and synthetic resins,antigens, and mixtures thereof. The stimulator compounds could includeother advanced glycosylation endproducts that may be prepared from thereaction between sugars and other macromolecules, and monokines whichstimulate phagocytic cells to increase their activity toward advancedglycosylation endproducts.

Accordingly, the stimulator compound may comprise the compound FFI boundto a protein such as albumin. Alternately, the stimulator compound maycomprise a synthetically derived advanced glycosylation endproduct whichis prepared, for example, by the reaction of glucose orglucose-6-phosphate with albumin. This reaction product can be usedalone or with a carrier in the same fashion as the FFI-albumin complex.

A monokine that functions as a stimulator compound comprises the proteinknown as Tumor Necrosis Factor (TNF) and its variant discovered andisolated by one of the inventors herein and named "cachectin". Thematerial may be administered alone or in conjunction with otherstimulator compounds.

In addition, the stimulator compounds of the present invention may beadministered in conjunction with materials identified hereinafter as"co-stimulatory agents". The coadministration of the stimulator compoundwith the co-stimulatory agents has been found to potentiate the activityof the former. Suitable co-stimulatory agents include monokines such asInterleukin-1 (IL-1) and gamma-interferon.

A further alternative embodiment of the method of the present inventionand one which may be practiced independently or conjointly with theabove recited method, is the ex vivo treatment of the phagocytic cellsto expose them to the stimulator compounds. For example, a patient maybe given an extracorporeal blood treatment in which blood is divertedout of the body from the arterial and venous system and is directedthrough a device which contains stimulator compounds and/orco-stimulatory agents which are suitably positioned to come in contactwith the phagocytic cells within the blood. The stimulator compoundsand/or co-stimulatory agents may be immobilized or may be allowed toenter the flow of the body fluid.

In the instance where the method comprises the in vivo administration ofthe stimulator compound and/or stimulatory agents, such administrationmay be accomplished by known techniques, including oral techniques andparenteral techniques such as intradermal, subcutaneous, intravenous, orintraperitonal injection, catheterization or other conventional means.The stimulator compounds or mixtures of them may be prepared in suitablepharmaceutical compositions for such administration.

In a further aspect of the present invention, phagocytic cells may bestimulated to increase their ability to recognize and remove targetmacromolecules by adjustment of the insulin level in the body fluid. Inparticular, artificial reduction of insulin levels may be achieved bydietary manipulation and/or by the use of pancreatic beta-cellsuppression, conducted alone or in combination with the administrationof the agents discussed above. Thus, the additional method exerts apositive effect on the activity of the phagocytic cells and promotes theincreased uptake and elimination of advanced glycosylation endproductsin accordance with the present invention.

In addition, the present invention relates to various therapeuticmethods, for the treatment of the adverse sequelae of the build-up ofadvanced glycosylation endproducts in the body. In particular,pathologies such as age related or diabetes related hardening of thearteries, skin wrinkling, arterial blockage and diabetic retinal andrenal damage are all the result of the excessive build-up or trappingthat occurs as the presence of advanced glycosylation endproductsincreases. Accordingly, a therapeutic method in accordance with thepresent invention generally seeking to avert such pathologiescontemplates the administration of the agents of the present inventioneither directly or in suitable pharmaceutical compositions to stimulatethe phagocytic cells to remove advanced glycosylation endproducts fromthe body with greater speed and efficiency, and to thereby avert theonset of the pathologies recited herein. Specific administrativeprotocols may vary and would be determined upon the specific instructionof qualified medical or veterinary practitioners.

The present invention method has particular application as the Maillardprocess acutely affects several of the significant protein masses in thebody, among them collagen, elastin, lens proteins, and the kidneyglomerular basement membranes. These proteins deteriorate both with age(hence the application of the term "protein aging") and as the result ofprolonged exposure to blood sugar and AGE formation. Consequently, theenhanced ability to remove glycosylation endproducts from the animal'ssystem carries the promise of favorably treating the significant adverseeffects of numerous pathologies including diabetes and of course,improving the quality and perhaps duration of animal life.

A further therapeutic application of the present invention lies in thearea of immunology. In particular, advanced glycosylation endproductsgenerally, and FFI in particular, may be coupled to antigens, andphagocyctic cells may then be exposed to this coupled complex to promotethe uptake and digestion of this coupled complex by such cells. Thephagocytic cells would then present the digested complex to the immunesystem of the animal of origin to elicit the development of antibodiesto the particular antigen. In this manner, antigens that might nototherwise elicit an immunologically significant response could be mademore immunologically reactive to develop effective defenses to theantigen. The foregoing method could be practiced in vivo or ex vivo, as,for example, the coupled complex including the advanced glycosylationendproduct or FFI could either be introduced into the body of theanimal, or phagocytic cells could be isolated from the animal inextracorporeal fashion and thereby contacted with the coupled complex,after which the phagocytic cells with their increased receptors could bereintroduced into the animal's body to appropriately stimulate theimmune system to develop antibodies.

A variant to the foregoing protocol contemplates the stimulation of thephagocytic cells to take direct action against the antigen. In thisprocedure, an antibody specific to the target antigen is labeled with orcoupled to an advanced glycosylation endproduct such as FFI, and thelabeled/coupled material is then introduced in vivo to promote thearousal of the activity of the phagocytes toward the material. Thelabeled/coupled material would bind to the target antigen and theresulting complex would be attacked by the phagocytes that recognize theAGE or FFI. Attack would occur either directly or by the secretion of amonokine such as cachectin to cause the necrosis of the antigen. Thephagocytes could be preliminary activated by in vivo or ex vivo means asdescribed earlier. This protocol, like the one described above,possesses particular utility as a possible treatment for Acquired ImmuneDeficiency Syndrome (AIDS) and other tumorous or viral infections.

In addition, the present invention contemplates certain diagnosticapplications, including the development of an assay system for screeningof potential drugs effective to act as agents to stimulate the activityof particular phagocytic cells against advanced glycosylationendproducts. In one instance, a prospective test drug could beadministered to a macrophage sample to determine its stimulatory effect,with control samples receiving, respectively, a known stimulatorcompound such as those listed above, and no stimulation whatsoever.Further, a particular phagocytic cell sample could be investigated todetermine the agents from among those known that are most effective instimulating such cellular activity, if such stimulation is possible, bythe inoculation of a series of identical sample colonies with various ofthe known agents recited above, with such agents being appropriatelylabeled by a radioactive indicator or otherwise, to chart the activityor progress in stimulation, by the uptake of such agents by theparticular cellular colony. In such manner, the colony exhibiting thegreatest uptake and disposal of labeled advanced glycosylationendproducts would identify the corresponding agent that is mosteffective in this stimulatory capacity.

By the above diagnostic techniques, cellular colonies capable ofstimulation could be determined, and in the instance where suchcapability is in evidence, the colonies could be further examined todetermine whether any discrimination in particular agent capable ofachieving such stimulation is in evidence.

Additional diagnostic applications relate to the study of a variety ofparameters attending the recognition and removal of advancedglycosylation endproducts by phagocytic cells and the significance ofthis phenomenon in relation to the functioning of the animal's system.In a first embodiment, phagocytic cells such as macrophages may beremoved from the animal's body and activated ex vivo by exposure toadvanced glycosylation endproducts. These activated phagocytic cells maythen be radiolabeled as with Technicium and thereafter reintroduced tothe animal and allowed to circulate through the animal's system, whilebeing radioimaged to note the final location of the cells. In thismanner, the location of concentrations of advanced glycosylationendproducts in the animal's body could be identified. This technique isparticularly useful in identifying undesirable concentrations ofadvanced glycosylation endproducts, such as atheromatous plaques. Insuch manner, the location of the systemic malfunction could beidentified.

Also, the condition of the system for the removal of advancedglycosylation endproducts from the body could be measured by thepreparation of radiolabeled advanced glycosylation endproducts and theadministration of these radiolabeled materials to the body to determinethe time required for their recognition, uptake and elimination. Suchmeasurement could then be compared against standard measurementsdetermined by testing normal systems under the same parameters. Theforegoing test could, for example, be performed as a test for diabetes,or other disorders that would adversely effect the operability of theAGE removal system of the body. Alternately, this method may likewise bepracticed in extracorporeal fashion by removing phagocytic cells fromthe body and testing them for their efficiency and rate of operationex-vivo with radiolabeled advanced glycosylation endproducts.

A further diagnostic technique could measure the presence of pathologyas a function of the state of activation of the phagocytic cells in thebody of the animal under investigation. Thus, macrophage cells could beexposed to particular radiolabeled advanced glycosylation endproductsknown to be found in connection with certain pathologies, and the stateof stimulation of the phagocytic cells could then be observed, bycomparison against suitably developed norms, to determine whether thephagocytic cells are in a state of stimulation, and if so, as to theprobable source of such stimulation. Thus, the presence of particularAGEs in the body reflect correspondingly particular pathologies, and theobservation of the phagocytic cells and the determination of theirsensitivity and increased stimulation with respect to particularadvanced glycosylation endproducts would suggest the existence of aparticular pathology.

Accordingly, it is a principal object of the present invention toprovide a method for improved sequestration and removal of advancedglycosylation endproducts from animal systems.

It is a further object of the present invention to provide a method asaforesaid which is characterized by the stimulation of phagocytic cellsto increase their affinity and capability for the binding, uptake anddegradation of advanced glycosylation endproducts.

It is a yet further object of the present invention to provide agentscapable of stimulating phagocytic cells to bind, take up and degradeadvanced endproducts in the method as aforesaid.

It is a still further object of the present invention to providetherapeutic methods for treating the adverse consequences of proteinaging.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing description which proceedswith reference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the relative binding and uptake of red bloodcells modified with various agents, including one of the agents of thepresent invention.

FIG. 2 is a graph illustrating the competitive inhibition in red bloodcell binding caused by the introduction into a sample of an agent inaccordance with the present invention.

FIG. 3 illustrates the binding and uptake by monocytes of red bloodcells that have been modified by reaction with a variety of sugars.

FIG. 4 is a line graph illustrating the half-life of red blood cellsthat have been labeled and modified by association with an agent inaccordance with the present invention, as compared with a control.

FIG. 5 is a bar graph illustrating the comparative uptake anddegradation of advanced glycosylation endproducts by mouse macrophagesexposed to various stimulator compounds.

FIG. 6 is a bar graph illustrating data similar to that set forth inFIG. 5, with respect to one day old human monocytes.

FIG. 7 is a bar graph illustrating similar comparative experimentsconducted with human monocytes wherein the co-stimulatory agent gammainterferon was also added and tested.

FIG. 8 is a graph illustrating the effect of insulin on the bindingcapabilities of mouse macrophage cells, wherein a normal sample and analloxan induced diabetic macrophage sample were compared.

FIG. 9 is a graph similar to FIG. 8, illustrating a comparison between anormal or control sample of human macrophages with those of hypo- andhyperinsulinaemic diabetic macrophages as to the binding capability ofeach of the samples.

FIG. 10 is a graph similar to FIG. 9, making a comparison between thesame macrophage samples as to their ability to degrade advancedglycosylation endproducts.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, a composition and associatedmethods have been developed for enhancing the removal of advancedglycosylation endproducts in animals, to treat protein aging and therebyinhibit the adverse effects thereof. Specifically, phagocytic cells suchas monocytes and macrophage cells are exposed to one or more agents orstimulator compounds which enhance the ability of such phagocytic cellsto recognize, bind and degrade advanced glycosylation endproducts.

The present invention is predicated upon the observation that monocytesand macrophage cells have the ability to recognize, remove and degrademacromolecules that have undergone glucose-mediated damage and thus haveundergone advanced glycosylation endproduct formation. In particular, ithas been determined that monocytes and macrophages have a specificsurface receptor for the glucose-altered macromolecules that allows thecells to perform their recognition, removal and degrading functions withrespect thereto.

Previous work with respect to glucose-mediated changes in proteins hasdetermined that glucose reacts with such macromolecules and leads tochanges in their properties which interfere with their normal function.Glucose is known to react specifically with the amino groups of proteinswithout the aid of enzymes to cause them to become crosslinked or tocovalently bind to and thereby trap other proteins.

In particular, glucose may initially react with an amino group on aprotein and thereby form what is known as a reversible Schiff baseadduct. The adduct then rearranges to form a more stable, but stillreversible Amadori product. The Amadori product, which is an initiallyglycosylated protein may then undergo further reactions, which in oneinstance, include the reaction with a second glycosylated amino group ofa protein, whereby a crosslink is formed. The result of this reaction isthe conversion of the two glucose moieties and their amino groups into acompound identified previously by one of the inventors herein andreferred to earlier as FFI. This compound is part of a complex family ofadvanced glycosylation endproducts that exhibit yellow and fluorescentappearance. The presence of this compound has been confirmed by the invitro reaction of glucose and protein, whereby the resulting mass isobserved to become yellow to brown in color. These advancedglycosylation endproducts form simply from the reaction of sugars withproteins and other macromolecules, including DNA.

The Amadori product can also react with a second glucose, and theresulting doubly glycosylated derivative can then react directly withthe amino group of a non-glycosylated protein to form an advancedglycosylation endproduct (AGE) and to thereby link the proteinstogether. This phenomenon has been termed, "trapping" and has beendemonstrated in vitro by the reaction of collagen, a normally insoluble,structural protein, and low-density lipoprotein, which is a circulating,soluble protein, as well as collagen with albumin and IgG. See Brownlee,M., et al., DIABETES, Vol. 34, pp. 938-941 (1985); Brownlee, M., Pongor,S., and Cerami A., J. EXP. MED., Vol. 158, pp. 1739-1744 (1983).

Glucose mediated crosslinking and trapping of proteins is a normalprocess in the body which over time leads to pathology in many tissuesand organs. Internal crosslinking of proteins or crosslinking of twoadjacent proteins may change the mechanical properties of structuralproteins. Changes in the immunologic, enzymatic, physical and otherproteins as a result of crosslinking and trapping are also known. Forexample, it has been observed that the abnormally high levels of glucosein the blood of diabetics leads to an abnormal increase in the formationof crosslinks and trapped proteins, and it is postulated that this maybe responsible for the increased morbidity and mortality of the disease.

Trapping leads to an abnormal build up of proteins in abnormal locationswhich can lead to pathology. Examples of the pathology that can becaused by glucose-mediated crosslinking and trapping includes theattachment of lipoprotein and other plasma proteins to the walls ofcoronary arteries, and its consequent build up of proteins andcholesterol to cause arterial blockage and heart attacks. Similarly,crosslinking of collagen in the arterial wall can change the mechanicalproperties of the arterial wall by stiffening its structure and therebycausing circulatory problems. Thickening of basement membranes ofsmaller blood vessels in the body and in the kidney resulting fromtrapping and crosslinking leads to peripheral vascular disease andthickening of the kidney basement membrane with subsequent loss ofkidney function (Nephropathy). In addition, thickening of vessel wallsin the brain leads to reduced blood flow and can contribute to the onsetof senility.

Crosslinking of collagen and other macromolecules in the skin may leadto wrinkling and other changes, as well as charges in the diabetic eyeand particularly damage to the lens and to the vessels of the retina,which latter event leads to a loss of visual acuity and ultimately, toblindness. Finally, glucose is known to react with DNA, and experimentshave shown that AGE-DNA can be mutagenic in bacteria.

As noted earlier, phagocytic cells are capable of recognizing andremoving abnormal macromolecules by means of receptors on their surfaceswhich recognize specific chemical structures and bind to them. Once theabnormal macromolecule is recognized in this way, the phagocytic cellmay internalize the macromolecule or particle containing the abnormalmacromolecule and may then degrade it. In some instances, the phagocyticcell may in addition secrete enzymes and other factors to help degradethe molecule or particle extracellularly if it cannot be internalized.After the damaged protein is removed, new growth of normal tissue canensue, and normal function of the affected area may resume.

Phagocytic cells in the body comprise numerous types of white bloodcells. One type of white blood cell, the monocyte, is produced in thebone marrow, and circulates briefly in the blood and thereafter entersthe tissues where it becomes a macrophage. Exposure of the phagocyticcell either as a monocyte or a macrophage, to certain molecules canregulate the appearance on the surface of the cell of receptors forthese molecules.

Thus, the present invention is predicated on the discovery that thephagocytic cells including monocytes and macrophages can be modified byexposure to certain agents or stimulator compounds that potentiate thecapability of these cells with respect to their recognition and affinityfor, and capability to degrade advanced glycosylated endproducts. Inparticular, the exposure of these cells to certain stimulator compoundshas been found to increase the number of receptors developed on thesecells and to thereby increased the capacity and efficiency of thesecells with respect to the recognition and degradation of advancedglycosylation endproducts.

Accordingly the method of the present invention generally comprisesexposing the animal body to certain agents or stimulator compounds,which cause the body, and its phagocytic cells in particular to becomeactivated and to increase its recognition and removal of targetmacromolecules that have undergone advanced glycosylation.

Suitable stimulator compounds useful in the present invention comprisematerials including advanced glycosylation endproducts either naturallyor synthetically formed which may be employed alone or bound to acarrier.

Suitable stimulator compounds include the compound FFI bound to acarrier protein such as the protein albumin. The stimulator compound mayalso comprise a synthetically derived advanced glycosylation endproductwhich is prepared, for example, by the reaction of a protein or othermacromolecule with a sugar such as glucose, glucose-6-phosphate, orothers. This reaction product could be used alone or could be combinedwith a carrier in the same fashion as the FFI-albumin complex.

The carrier may be selected from the group consisting of carbohydrates,proteins, synthetic polypeptides, lipids, bio-compatible natural andsynthetic resins, antigens and mixtures thereof.

As used herein, the term "antigen" includes various invasive stimulithat may comprise or cause the onset of pathology or other organicdisability, such as protein and lipid fragments, bacteria, virusesand/or other organisms of similar origin and effect.

Stimulator compounds also include other monokines which stimulatephagocytic cells to increase their activities toward advancedglycosylation endproducts.

A particular monokine that functions as a stimulator compound comprisesthe protein known as Tumor Necrosis Factor (TNF) and its variantdiscovered and isolated by one of the inventors herein and named"cachectin". This material may be administered alone or in conjunctionwith other stimulator compounds.

In addition, the stimulator compounds of the present invention may beadministered in conjunction with materials identified hereinafter as"co-stimulatory agents". The coadministration of the stimulator compoundwith the co-stimulatory agents has been found to potentiate the activityof the former. Suitable co-stimulatory agents include monokines such asInterleukin-1 (IL-1) and gamma-interferon.

With regard to the preparation of the stimulator compound comprising FFIcoupled to a carrier molecule such as albumin, the synthetic compoundFFI-hexanoic acid may be used in its preparation. Thus, a water-solublecarbodiimide is used to attach the acid moiety of the FFI-hexanoic acidto an amino group on the protein. The conjugate, after purification, isused in vitro to stimulate macrophages. After incubation for 4 to 24hours, it can be shown that such macrophages will more actively bind,internalize, and degrade AGE-albumin.

Accordingly, the present invention includes various therapeutic methodsseeking to treat the adverse effects of the buildup of advancedglycosylation endproducts in animals. Such conditions as age- ordiabetes-related hardening of the arteries, skin wrinkling, arterialblockage and diabetic retinal and renal damage all result from theexcessive buildup or trapping that occurs as advanced glycosylationendproducts increase in quantity. Accordingly, a therapeutic methodseeking to avert pathologies caused at least in part by the accumulationof advanced glycosylation endproducts in the body comprises theadministration of the agents of the present invention either directly orin suitable pharmaceutical compositions to stimulate the body theincrease its activity toward the recognition and removal of suchadvanced glycosylation endproducts. In particular, the agents areadministered to stimulate the phagocytic cells in the body to increasetheir activity toward the recognition and removal of advancedglycosylation endproducts so that such removal occurs with greater speedand efficiency. Specific adminstrative protocols would vary and would bedetermined upon the instruction of qualified medical or veterinarypractitioners.

Accordingly, the present invention also includes suitable pharmaceuticalcompositions for use in the therapeutic methods of the invention,comprising the agents of the present invention prepared in a suitablepharmaceutically acceptable carrier. Such carriers are known and mayvary in composition and/or concentration depending upon the manner ofadministration, i.e. oral, parenteral, etc.

The present invention will be better understood from a consideration ofthe following illustrative examples and data, that confirm theactivities of the phagocytic cells and their relationship to thestimulator compounds discovered in accordance herewith.

EXAMPLE I

In the investigation which follows, the existence in vivo of theclearance system of advanced glycosylation endproducts was confirmed andstudied, and the principles of the present invention were established.

MATERIALS AND METHODS

RBC preparation: Human Blood (2.0 ml) from normal, healthy adultvolunteers was collected in heparinized tubes. Following removal ofplasma and buffy coat, the RBC's were washed four times with 10 vol ofCa²⁺ and Mg²⁺ free phosphate-buffered saline (PBS), pH 7.4 and wereresuspended in Dulbecco's modified Eagle medium.

Opsonized RBC preparation: 0.1 ml of a 15% RBC suspension from a D+donor was added to 0.5 ml of a high-titer anti-D serum and was incubatedat 37° C. for 30 minutes. Following incubation, cells were washed threetimes with PBS (GIBCO) and were resuspended in 3 ml of Dulbecco'smodified Eagle medium.

FFI-RBC preparation: The specific advanced glycosylation endproduct(AGE) [2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole]-hexanoic acid(FFI-HA) was prepared as described previously (Pongor et al., PNAS(1984) Vol. 81, pp. 2684-2688). In brief, furylglyoxal hydrate (10 mmol)in 3:1 dioxane/water was treated with 6-amino hexanoic acid (15 mmol)and triethylamine (15 mmol) and stirred at 25° C. for 1 hour. After theaddition of concentrated aqueous ammonia, the mixture was diluted with5% NaH₂ PO₄ and extracted with CH₂ Cl₂, washed with brine and filteredthrough activated carbon and MgSO₄. The crude product was purified bymedium pressure chromatography on silica gel yielding FFI-HA asstraw-colored flakes (m.p. 105°-106° C.). Freshly washed normal RBC'swere resuspended in PBS, with and without 10 mM water soluble1-cyclohexyl-3-[2-(4-morpholinyl)-ethyl]-carbodiimide (CMC) (Vlassara,et al., (1985), Vol. 82, pp. 5588-5592), to which FFI-HA was added atdifferent concentrations (10-100 μM). The mixtures were incubated undercontinuous mixing for 1 hour at room temperature. Parallel mixturescontaining RBC's alone in PBS or RBC's and carbodiimide (10 mM) weretreated identically as controls. After 3 washes the cells wereresuspended in 1 mM glycine in PBS and left to incubate for 30 minutes.Following three washings with PBS the cells were suspended in RPMI priorto phagocytosis assay, at an 100 excess ratio to monocytes.

Glycosylated-RBC preparation: In order to produce nonenzymaticallyglycosylated erythrocytes, glucose, glucose-6-phosphate, xylose andarabinose were added to freshly isolated normal human red cellssuspended in Dulbecco's modified Eagle medium at 100 mM concentrationsand incubated for 48 hours at room temperature. RBC suspensions withoutsugars added were used as control cells. Following the incubation, thecells were washed three times with PBS and were suspended in RPMI.

AGE-BSA was prepared by incubating bovine serum albumin (BSA) in 50 mMglucose at 37° C. for 6 weeks, in the presence of protease inhibitors(PMSF 1.5 mM, EDTA 0.5 mM) and antibiotics (penicillin 100 U/ml,gentamicin 40 mg/ml) (See, Vlassara et al., Supra.)

Human monocyte preparation: The buffy coat from 100 ml fresh human bloodwas diluted two-fold with saline 1 mM EDTA, pH 7.4, and mononuclearcells were separated from other elements of the blood by centrifugationon Ficoll-Paque gradients as described before (Gimelig-Meyling et al.(1950), J. IMMUN. METHODS, Vol. 33, p. 1). The mononuclear cells werewashed three times in cold RPMI 1640 (GIBCO) to remove platelets, andthe cells were suspended in RPMI made 10% in normal human serum. Toobtain suspensions of monocytes for use in these experiments, thePercoll purification method described previously was used(Gimelig-Meyling supra.). Percoll was brought to isotonicity by theaddition of 0.1 vol. of 10-X concentrated PBS. One ml of normal humanserum, 14.7 ml PBS, and 22 ml isotonic Percoll was mixed in sterile50-ml centrifuge tubes and centrifuged for 25 minutes at 18,000 rpm at5° C. in Sorvall SS-34 rotor. Five ml of the mononuclear suspension werelayered on the resulting gradients and the tubes were centrifuged at1,500 g for 25 minutes at 5° C. in a swinging bucket rotor. Theresulting bands were detected by light scattering and band I,corresponding to monocytes was transferred and cultured in screw capTeflon jars (Savillex, Minnetonka, Minn.), at 10⁶ /ml in RPMI with 12.5%human serum at 37° C. in 5% CO₂ for 5-7 days. Cell viability wasassessed by trypan blue exclusion (Gibco Lab.) and plating efficiency ofphagocytes on a tissue-culture plastic surface was measured by ahemocytometer before and after attachment. When ready for use, aliquotsof 10⁶ monocytes were plated in sterile petri dishes containingprecleaned round coverslips and incubated for 2 hours at 37° C. in 5%CO₂.

Phagocytosis assay: Before the addition of RBC's the monocyte cultureswere washed twice with RPMI 1640. The various red cell suspensions wereadded to each well at a 100 fold excess to the monocytes and wereallowed to incubate at 37° C. and 5% CO₂ for up to 2 hours. At thatpoint the coverslips were removed from the wells, washed three timeswith RPMI to remove nonadherent material, placed into clean wells andfixed with 1.25% glutaraldehyde in PBS for 30 minutes. To differentiatesurface-attached from ingested erythrocytes, a hypotonic solution (PBSdiluted 1:4 in H₂ O) was added to selected wells for 10 seconds,followed by the addition of fixative. To read the assay, duplicatecoverslips were counted using 40X phase microscopy, At least 300monocytes from three or more randomly selected fields were counted perwell. The data were expressed as the number of positive monocytes(monocytes with erythrocytes attached or ingested) per 100 monocytes(percent binding). Ingestion index was also determined as the number ofingested or adhered red cells per positive monocyte (Bianco et al.(1976) J. EXP. MED., Vol. 144, p. 1531).

FFI-RBC 1/2 life assay: Balb/c inbred mice were bled by cardiac punctureyielding approximately 2.0 ml of blood. Red cells were washed with largevolumes of divalent cation-free PBS and coupled with FFI-HA as describedabove in the presence of 10 mM CMC. In addition, RBC's incubated in CMCalone or PBS alone were used as controls. Subsequently all cellsuspensions were labeled with ⁵¹ Cr by adding 0.2 mCi Na₂ [⁵¹ Cr]O₄ to 2ml of 50% packed RBC in RPM 1-1640 medium for 1 hour at 37° C. Thelabeled cells were washed at least four times to remove unbound isotope.Twelve Balb/c mice were then injected intravenously with 200 μl RBCsuspension. Each sample was administered in three Balb/c mice. Atappropriate time intervals the mice were bled (0.2 ml) and radioactivitylevels were measured by counting.

RESULTS

Maximum binding of red cells was observed on Day-7 of monocyteincubation in vitro. Maximum binding and endocytosis of FFI-RBC wascomplete within 30-45 minutes while opsonized cells were maximally boundwithin 15 minutes. At the end of one hour incubation of FFI-coupledRBC's with cultured human monocytes, per cent phagocytosis andphagocytic index were estimated. As shown in FIG. 1, %erythrophagocytosis of FFI-modified red cells (55%) and IgG-coated redcells (70%) were significantly higher than that of control PBS-treatedcells (4%). Similarly the phagocytic index of FFI-treated RBC's wassignificantly elevated (3,4) as compared to normal controls (1,2).

In order to establish the specificity of the interaction of FFI-RBC'swith the human monocytes, competition experiments were carried out inwhich binding and ingestion of red cells was observed in the absence andpresence of AGE-BSA, prepared as described in Methods (Vlassara et al.,supra.). As shown in FIG. 2, the addition of AGE-BSA at concentrationsof 500 μg/ml inhibited the FFI-RBC binding by more than 70% of thecontrol. In contrast, AGE-BSA did not inhibit opsonized PBS-treated redcells, even at maximal concentrations (1 mg/ml). These data suggestedthat FFI-modified red cells were recognized and bound specifically bythe monocyte AGE-binding site.

Advanced glycosylation endproduct (AGE) formation was then induced onred cell surfaces by 48 hour in Duibecco's modified Eagle mediumcontaining different sugars, such as glucose, glucose-6-phosphate,xylose and arabinose, at 100 mM concentrations, at room temperature. Atthe end of this period, the media demonstrated no evidence of celllysis, and red cells themselves appeared microscopicallyindistinguishable from the controls. As demonstrated by RIA (the methodof Chang et al., (1985) J. BIOL. CHEM., Vol. 260, pp. 7970-7974), allcells incubated in sugar underwent formation of a significant amount ofadvanced glycosylation endproducts on their cell membrane, as comparedto the controls. At this point RBC's from all groups were subjected tonormal human monocyte phagocytosis assay. As indicated in FIG. 3,glucose-incubated RBC's showed a 15% binding and ingestion andG6P-treated RBC's at 26%, as compared to 6% binding and ingestion ofcontrol PBS-RBC's. Much level of binding were noted with red cellspreincubated with xylose and arabinose (7.5% each).

In order to determine whether autologous red blood cells modified by anAGE such as FFI-HA can be recognized by macrophages in vivo and beremoved from the circulation faster than the non-modified cells,presumably by the same AGE-receptor present on hepatic and splenicmacrophages, Balb/c inbred mouse red cells were treated either withFFI-HA, as described in Methods or PBS alone for one hour at roomtemperature. As an addititonal control, mouse cells were treated withcarbodiimide alone (10 mM). Following, ⁵¹ Chromium-labeling all threegroups of cells were reinjected intravenously into syngenetic mice anderythrocyte radioactivity was monitored for 20 days. An approximately 9%of initial radioactivity recovered from all groups within the 1 hour ofthe injection was recovered in the serum fraction, presumably due totraumatic hemolysis caused by the ex vivo handling since it was reducedto 0.4% within the first 24 hours. As shown in FIG. 4, the half-life ofFFI-treated red cells was reduced to 7 days, as compared to the controluntreated cell half-life of approximately 20 days. Similarly, cellstreated with carbodiimide alone had nearly normal in vivo course.

DISCUSSION

The above tests extend previous observations on the recognition ofadvanced glycosylation endproducts (AGE) by a specificmonocyte/macrophage receptor, and present evidence that such adductsonce attached chemically or formed in vitro on the surface of intacthuman cells can induce cell binding and ingestion by normal humanmonocytes. It is demonstrated that the presence of AGE on the cellsurface in significant amounts over the unmodified cells leads to theirshortened survival in vivo, presumably due to the more rapid removal ofthese AGE-cells by the splenic and hepatic phagocytic cells. Themechanism of recognition and removal of such modified cells appears tobe mediated via the specific macrophage AGE-receptor, as shown by thecompetitive inhibition only of AGE-red cell binding in the presence oflarge excess of AGE-BSA (Vlassara et al., supra.).

With age, nonenzymatically glycosylated proteins normally undergofurther modifications and rearrangements leading to the formation ofincreasing amounts of AGE's, as was shown in normal human dura collagen(Monnier et al. (1984) PNAS, Vol. 81, pp. 583-587). One specificendproduct, FFI, has been identified in vivo on normal human serumalbumin and globin (Pongor et al., (1984) PNAS, Vol. 81, p. 2684). TheAGE-receptor recognition increases with the amount of AGE, and cantherefore preferentially recognize a time dependent signal for theremoval of senescent macromolecules (Vlassara et al. supra.).

Nonenzymatic glycosylation of erythrocyte membrane proteins has beenshown previously (Miller et al., (1980) J. CLIN INVEST. Vol. 65, pp.896-901) to occur primarily at the lysine residues of all the majorprotein bands without distinction and is enhanced in diabetes, while itis decreased in hemolytic anemia. These findings have suggested that themodification of membrane proteins, other than by blood glucose, dependsnot only on glucose concentration, but also on erythrocyte age. Theforegoing experiments demonstrate that AGE is present on normal intactred blood cells, which may be responsible in part for daily removal oferhythrocytes from the circulation by the monocyte/macrophageAGE-receptor. Further, AGE accumulation can be accelerated by exposureto high glucose levels, which in turn leads to increased monocyteAGE-receptor binding and ingestion of glucose-modified red cells, andthis may play a role in the moderately shortened erythrocyte survival indiabetics (Peterson, C. M., et al., (1977) ANN. INT. MED., Vol. 86, pp.425-429).

The difference in erythrocyte binding between glucose andglucose-6-phosphate--treated cells is probably due to the higher amountof AGE formed, owing to the presence of a higher percentage of morereactive open ring structures in the incubation mixture in the case ofglucose-6-phosphate. Suprisingly, even through xylose and L-arabinoseboth can react faster with protein amino groups than glucose, as shownby the amount of AGE formed, they did not induce binding and uptakeabove the normal control cells. This phenomenon warrants furtherinvestigation, however it is probably due to formation of glycosylationproducts which are not recognized by the AGE or any other monocytereceptor.

EXAMPLE II

The following series of experiments were performed to measure theability of agents to stimulate phagocytic cells to stimulate uptake anddegradation of endproducts (AGEs).

Accordingly, a number of AGEs were prepared using the same procedure asdisclosed in Example I, above. Thus, FFI-HA was prepared as describedand quantities were bound to both human and bovine albumin. A watersoluble carboiimide was used to attach the acid moiety of the FFI-HA toan amino group on the protein. After preparation, the conjugate waspurified and then used in vitro to stimulate microphages, by incubationfor from 4 to 24 hours. The AGEs that were to be observed for uptake anddegradation were appropriately radiolabeled so that they could betraced. Thereafter, the stimulated macrophages were tested by exposureto the radiolabeled AGEs following exposure to various agents to measurethe effect that these agents had on the ability of the macrophages totake up and degrade the labeled AGEs. The above procedures and thestudies that follow conform to the protocol employed by Vlassara et al.,supra.

Thus, FIG. 6, Part I, shows the uptake of AGE-BSA by human monocytesafter stimulation with certain agents in the absence ofgamma-interferon. The FFI-human albumin at 0.1 and 0.2 mg/ml had astimulatory effect on the uptake system (bars E and F, respectively) ascompared to the control (bar A). FFI-bovine albumin is also stimulatory(bars E', F', and G'). Gamma-interferon alone (bar C), orlipopolysaccharide alone (bar B) or Interleukin-1 alone (bar W) have nostimulatory effect.

FIG. 6, Part II, shows the degradation of AGE-BSA by human monocytes inthe presence of these agents, in the absence of gamma-interferon. TheFFI-BSA preparations are slightly stimulatory (bar Y). In FIG. 7, Part1, the effect of stimulation on the uptake of AGE-BSA by the same agentsin the presence of 10 micrograms/ml of human gamma-interferon is shown.As can be seen, gamma-interferon greatly potentiates (up to 8-fold) thestimulation by 0.01, 0.02, and 0.05 mg/ml of FFI-BSA (bars N, O, and P).Degradation is also enhanced in the presence of these agents plusgamma-interferon (FIG. 7, bar V).

It has also been demonstrated that monocyte or macrophage cells can alsobe stimulated by AGE-carrier molecules which result in cells withenhanced ability to bind, internalize and degrade other AGE-molecules.AGE-carrier molecules are made, for example, from the reaction ofglucose or glucose-6-phosphate with albumin. After purification of thereaction product, the AGE-albumin uptake of AGE-macromoleculesdemonstrated as in (A) above. AGE-BSA (prepared from the incubation ofglucose-6-phosphate with albumin for 6-8 weeks) at 0.1 mg/ml has astimulatory effect on AGE-BSA uptake by human monocytes (FIG. 6, barAA), and shows a slight stimulation at higher concentrations (bars BBand CC). FIG. 7, bar S, shows that in the presence of gamma-interferon,0.5 mg/ml of AGE-BSA made from glucose-6-phosphate, greatly stimulatesuptake of AGE-BSA by macrophages. The lower concentrations show a slightstimulatory effect (bars Q and R).

Degradation of AGE-BSA by human monocytes is also stimulated by AGE-BSAin the presence of gamma-interferon (FIG. 7, bar X). Without interferon,stimulation is slight (FIG. 6, bar Z).

Mouse peritoneal macrophages show increased uptake of AGE-BSA whenstimulated with AGE-BSA prepared from glucose-6-phosphate (FIG. 5, barGG). Degradation is also increased (FIG. 5, bar MN).

As discussed earlier, phagocytic cells such as macrophages may bestimulated to become activated and remove AGE-macromolecules aftertreating the macrophages with a monokine such as with cachectin(synonym=tumor necrosis factor). The structure and properties ofcachectin have been previously elucidated by one of the inventorsherein. It has been determined that cachectin as a concentration of 20ng per milliliter of culture fluid stimulates macrophages to express theAGE-receptor and to increase binding, uptake and degradation ofAGE-macromolecules. Preincubation of mouse macrophages for 24 hours with20 ng/ml of cachectin/TNF (without gamma-interferon) followed byincubation with AGE-BSA results in a 4-fold increase in AGE-BSA (madefrom glucoe reacted with albumin uptake (FIG. 5, bar FF). Degradation isalso increased by 50% of the control (bar MM).

Similarly, preincubation of 1-day old human monocytes for 24 to 48 hourswith cachectin without gamma-interferon leads to almost doubling ofuptake and degradation of AGE-BSA (FIG. 6, bar D). Degradation is alsoincreased (bar 1).

The above examples of agents which stimulate macrophages to increase theability to internalize and degrade AGEs should not be restrictive. Otheragents include additional synthetic specific AGEs linked to carriermolecules, other AGEs made from the reaction of sugars withmacromolecules, and other monokines which stimulate macrophages toincrease their activity toward AGEs.

The method also includes the coadministration of these agents and one ormore co-stimultatory agent such as Interleukin-1 and gamma interferon toachieve an even greater stimulation of the body's AGE removal system.

In a further embodiment of the present invention, macrophages could betreated ex vivo with these agents and returned to the body. For example,an extracorporeal blood treatment may be performed in which indwellinglines are placed in a patient's arterial and venous system, and blood istaken from the body, passed through a device and then returned to thepatient. The device is contemplated to contain agents which by contactwith or exposure to monocytes/macrophages will stimulate them toincrease the activity of the AGE removal system. Further, the agents mayeither be immobilized or may be capable of entering the blood flow. Suchan extracorporeal process may be performed alone or jointly with theadministration to the patient of other agents to enhance the process,such as the gamma-interferon described above.

An additional aspect of the present invention herein relates to theobservation of the effects of insulin on the macrophage AGE clearancesystem. It has been found that animals with lower than normal levels ofinsulin in the blood have an enhanced macrophage AGE clearance system.This has been demonstrated in both experimental animals in whichinsulin-producing pancreas cells are destroyed by injection of theanimal with alloxan, or in genetically diabetic animals which have lowinsulin levels. In both groups, blood glucose levels are higher thannormal. As shown in FIG. 8, animals with experimentally-induced diabetes(using alloxan) and resultant low serum insulin levels (25±6 μU/ml) hada two-fold greater activity of binding of AGE-BSA to macrophages ascompared with normal animals (serum insulin of 74±28 μU/ml). In FIG. 9,it is apparent that C57BL/KsJ, db/db mice, with genetically low insulinlevels (82±2 μU/ml) have a greater degree of AGE-BSA binding tomacrophages than control mice.

In contrast, animals with high levels of insulin in the blood, such asgenetic hyperinsulinaemic animals (C57BL6, db/db serum insulin>300μU/ml, FIG. 9), have a reduction of about 50% of the activity of bindingof AGE-BSA to macrophages compared to normal animals. This suppressionof the removal system would have a negative effect since clearance ofAGE-macromolecules would be decreased. Degradation of AGE (FIG. 10)shows the same relationship to insulin levels.

It is important to note that both the hypoinsulinaemic andhyperinsulinaemic animals had equally abnormal elevations of glucose inthe blood.

The effect of insulin on other macrophage receptors is known, however,that is the first report of the role of insulin in regulating the AGEreceptor. Because insulin regulates the glucose level in the blood, andglucose is responsible for the production of AGEs which are removed bymacrophages, this novel finding suggests an actual correlation betweenthese three phenomena.

An additional observation derived from the present invention, and onewhich proposes a possible mechanism for the effects of the presentmethod is that macrophages, on stimulation with FFI-carrier proteins orAGE-proteins, will secrete cachectin into the surrounding fluid.Macrophages are known to produce cachectin in response to certainstimuli, but this is the first report of the monokine being produced inresponse to FFI and AGE-proteins. Since these moieties exist in thebody, production of cachectin may occur during recognition,internalization, or degradation of AGEs. Since cachectin is known toincrease the removal system, this observation suggests that cachectinmay be involved in an amplification phenomenon to signal othermacrophages to increase activity of the removal system. The secretion ofcachectin by cells exposed to AGEs may also bring about other effects oncells.

Accordingly, one of the therapeutic methods of the present inventioncomprises providing cachectin or a derivative of cachectin in amountssufficient to stimulate the removal system. The exact quantities ofcachectin to be administered may vary, and the amounts employed in theexperiments with cachectin set forth herein are representative.Naturally, specific amounts would be determined by the attendingphysical or veterinarian administering the treatment.

A further therapeutic application of the present invention lies in thearea of immunology. In particular, the recognition and degradation ofadvanced glycosylation endproducts, such as FFI by phagocytic cellsfacilitates the introduction to those cells of certain antigens againstwhich it is desired to raise an immunity. Accordingly, the advancedglycosylation endproduct or FFI could be coupled to an antigen in muchthe same fashion as disclosed herein for the coupling to any othercarrier. Thereafter, the particular phagocytic cells that it is desiredto stimulate could be exposed to the coupled complex whereupon the cellswould recognize and degrade the latter, and would concomitantly developspecific receptors therefor. These activated phagocytes could then beintroduced to the immune system of the animal and would promote thedevelopment by the immune system of antibodies to the initial antigen.

The above method may be practiced ex vivo or in vivo. If ex vivo, themethod would include the initial removal of the phagocytes from the bodyand their exposure to the coupled complex to develop their sensitivityto the particular antigen. Thereafter a sample of cells of the animalthat is known to be responsible for raising antibodies and that wouldhave been similarly isolated and removed from the animal, or cells fromother sources that may perform the same function, would be exposed tothe activated phagocytes for a period of time sufficient to enableantibodies to be raised to the antigen of interest. The antibodies couldbe administered to animals to avert or alleviate the adverse effects ofany pathology that might be caused by the invasion of the antigen.

Alternately the ex vivo activated phagocytes could be utilized as avaccine to inoculate animals against the antigen and to thereby promotethe in vivo development of antibodies thereto.

An in vivo protocol contemplates the preparation of the coupled complexbetween the AGE/FFI and the antigen and the administration of thiscoupled complex directly to the animal to promote the in vivo activationof phagocytes and the subsequent development of antibodies by theanimal's immune system.

A further alternative contemplates the formation of a mixture ratherthan a coupled complex between the AGE/FFI and the antigen. This mixturecould be utilized in place of the coupled complex in the aboveprotocols.

A particular implementation of the ex vivo embodiment of theimmunological protocol could include the immobilization of either thecoupled complex or the phagocytic cells during all or part of thepractice of the particular method. Thus, for example, either the cellsor the coupled complex could be immobilized and the unbound materialthen circulated therepast to achieve activation. If it is desired toadminister the bound material to the animal, it could be released fromthe substrate after activation by known techniques.

A further therapeutic protocol is suggested by the foregoingimmunological protocols, which is based upon the binding, labeling orother combination of the AGE/FFI in this instance to a particularantigen as defined herein. Thus, the AGE/FFI could be combined with anantibody specific to the particular antigen, and the resultingassociated mixture or combined material then placed in contact with thephagocytic cells of the animal/human host to stimulate such cells torecognize and attack the antigen. In such instance, the manner by whichcontact between the combined material and the phagocytes occurs mayvary.

Thereafter, a quantity of the combined material may be introduced invivo to bind with the target antigen. The stimulated phagocytes wouldthen be introduced or stimulation of the phagocytes would take placeconcurrently upon the in vivo introduction of the combined material,whereupon the phagocytes would attack and destroy/degrade the complexformed by the combined material and the target antigen, either directlyor by means of the secretion of the monokine cachectin/TNF. Regardlessof the exact mechanism of final action, the phagocytes would actspecifically against the target antigen because of their recognition ofthe particular AGE/FFI now associated therewith by means of theantibody.

The foregoing therapeutic method may vary as to dosage, manner ofadministration and periodicity, depending upon thee particular host andthe attending pathological condition, and is subject to adjustment bythe trained physician or veterinarian. This therapy offers a potentiallyeffective avenue of treatment for patients suffering from AcquiredImmune Deficiency Syndrome (AIDS) in view of the inability ofconventional therapies to alleviate the condition.

All of the above immunological and therapeutic protocols could includethe coadministration of one or more of the co-stimulatory agents in themanner set forth earlier herein to potentiate where possible theefficacy of such protocols. The present invention accordingly extends tosuch variations in its practice.

As mentioned earlier, the present invention contemplates numerousdiagnostic applications. In a first application, an assay system may bedeveloped for screening potential drugs effective to act as agents tostimulate the activity of specific phagocytes against advancedglycosylation endproducts. Accordingly, a prospective test drug could beadministered to a macrophage sample to determine its stimulatory effectand the macrophage sample after incubation with the test drug could beincubated with a fluid or tissue sample having a quantity ofappropriately labeled advanced glycosylation endproducts presenttherein, so that comparative uptake and degradation studies could thenbe made. In this assay, plural macrophage colonies could be initiallyincubated with various of the known agents, so that comparative testingof the prospective drug could take place with greater specificity.

An alternate diagnostic protocol contemplates the investigation ofparticular phagocytes to determine which of the known agents are mosteffective in stimulating cellular activity against advancedglycosylation endproducts. Thus, in similar fashion to the protocoldescribed above, plural comparable quantities of a particular phagocyticcell colony could be isolated and incubated with several different knownagents, and thereafter incubated with identical corresponding samplescontaining appropriately labeled advanced glycosylation endproducts, sothat a comparison of the extent of activation of the phagocytic cellscould then be made, and the agent most effective in stimulating thecellular colony thereby identified. In such manner, the colonyexhibiting the greatest uptake and disposal of labeled advancedglycosylation endproducts would correspondingly identify the agent thatis most effective.

A further diagnostic utility is predicated upon the study of the variousparameters that attend the recognition and removal of advancedglycosylation endproducts and their significance in relation to theoperation of animal body systems. In a first embodiment, phagocyticcells such as monocytes and macrophages are removed from the animal'sbody and are activated ex vivo by exposure to the agents of the presentinvention. This may be accomplished by an extracorporeal shut as hasbeen described earlier on herein. After such activation, the phagocyticcells, instead of being returned immediately to the body, areappropriately radiolabeled, as with Technicium, and are then returned tothe body, whereupon the animal may undergo radioimaging to note thecourse of travel of the phagocytes and to thereby determine the locationof the concentrations of advanced glycosylation endproducts in the body.In this manner, undesirable concentrations of advanced glycosylationendproducts, such as atheromatous plaques could be discovered, and theclinical significance of these concentrations accordingly assessed. Thisprocedure would provide additional vital information in attempting toassess the pathological state of the animal or patient.

A further diagnostic application likewise seeking to provide vitalinformation regarding pathological status contemplates the preparationof radiolabeled agents and their incubation or contact with phagocytesfrom a particular animal, and the subsequent measurement of the elapsedtime before the labeled agents are recognized and degraded. Such timemeasurements could be compared against standard measurements determinedby testing cells taken from normal animals or patients that were testedunder the same protocol. This data could then identify the existence ofpathologies such as diabetes, or the others mentioned earlier herein, orother disorders that may adversely affect the operability of the AGEremoval system of the body. This diagnostic procedure can be performedin vivo by administering the labeled agents to the animal or patient, oralternatively, could be conducted ex vivo by the isolation of phagocyticcells of the animal or patient outside the body and the incubation ofthese cells with the labeled agents.

An additional diagnostic technique contemplates a further qualitativeanalysis of the animal's body. In this procedure particular pathologiesmay be identified by a method wherein the activity of the phagocycticcells of the animal could be measured and compared against theactivities found with normal cells. In such instance, for example,radiolabeled agents could be introduced to the body, or phagocyticcellular colonies from the body could be isolated and incubated with theradiolabeled agents, and the level of activity of the phagocytic cellscould then be measured with greater specificity as to particularadvanced glycosylation endproducts. This is possible because of thespecific nature of the cellular receptors that develop on the phagocyticcells as to particular advanced glycosylation endproducts. Thus, themacrophage or monocytes having greatest involvement with the removal ofadvanced glycosylation endproducts that would otherwise developatheromatous plaques, could be isolated and tested for their activity,and if their activity appears to be abnormally increased, thedevelopment of such plaques may be indicated. Similar testing protocolscould be employed to determine the existence of diabetic aging and thelike.

Thus, the specific advanced glycosylation endproducts associated withparticular conditions would elicit responses from the phagocytes thatwould suggest any abnormal accumulation of these advanced glycosylationendproducts in the body system. The presence of particular AGEs in thebody reflect correspondingly particular pathologies, and the foregoingobservation of the phagocytes and the determination of their sensitivityand increased stimulation with respect to particular advancedglycosylation endproducts would suggest the existence of a particularpathology.

This invention may be embodied in other forms or carried out in otherways without departing from the spririt or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended Claims, and all changes which comewithin the meaning and range of the equivalency are intended to beembraced therein.

What is claimed is:
 1. A method for averting the adverse sequelae of theaccumulation of advanced glycosylation endproducts in the body of ananimal, comprising introducing into said body an effective amount of anagent capable of causing said body to increase its activity ofrecognizing and removing macromolecules that have undergone advancedglycosylation selected from the group consisting of an advancedglycosylation endproduct, an advanced glycosylation endproduct bound toa carrier, .[.the fluorescent chromophore2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole bound to a carrier.]. amonokine that stimulates the phagocytic cells in said body to increasesaid recognizing and removing activity toward said macromolecules, andmixtures thereof.
 2. The method of claim 1 comprising introducing anagent capable of causing said body to increase its activity orrecognizing and removing macromolecules that have undergone advancedglycosylation in combination with a co-stimulatory agent whichpotentiates the activity of said agent.
 3. The method of claim 2 whereinsaid advanced glycosylation endproduct comprises the reaction product ofa proteinaceous macromolecule and a sugar.
 4. The method of claim 2wherein said advanced glycosylation endproduct is selected from thegroup consisting of the reaction product of albumin and glucose, thereaction product of albumin and glucose-6-phosphate, .Iadd.thefluorescent chromophore 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole boundto a carrier, .Iaddend.and mixtures thereof.
 5. The method of claim 2wherein said carrier is selected from the group consisting ofcarbohydrates, proteins, lipids, synthetic polypeptides, biocompatiblenatural and synthetic resins, antigens, and mixtures thereof.
 6. Themethod of claim 2 wherein said monokine comprises the polypeptideidentified as tumor necrosis factor.
 7. The method of claim 2 whereinsaid co-stimulatory agent is selected from the group consisting ofInterleukin-1 and gamma-interferon.
 8. The method of claim 1 whereinsaid agent is administered by parenteral means.
 9. The method of claim 8wherein said parenteral means comprises injection.
 10. The method ofclaim 8 wherein said parenteral means comprises catheterization.
 11. Themethod of claim 2 wherein said agent is administered by oral means. 12.The method of claim 2 wherein said agent is prepared as a pharmaceuticalcomposition, with a pharmaceutically acceptable carrier.
 13. The methodof claim 1 wherein said agent is administered both parenterally andextracorporeally to the body fluid of said animal body.
 14. The methodof claim 1 wherein said body contains a quantity of insulin, and saidmethod includes the treatment of said body to lower the available activeinsulin level thereof.
 15. A method for averting the adverse sequelae ofthe accumulation of advanced glycosylation endproducts in the body of ananimal, comprising:isolating a quantity of phagocytic cells from saidanimal; treating the phagocytic cells so isolated with an agent capableof causing said phagocytic cells to increase their activity ofrecognizing and removing macromolecules that have undergone advancedglycosylation selected from the group consisting of an advancedglycosylation endproduct, an advanced glycosylation endproduct bound toa carrier, .[.the fluorescent chromophore2-(2-furoyl)4(5)-(2-furanyl)-1H-imidazole bound to a carrier.]. amonokine that stimulates the body to increase said recognizing andremoving activity toward said macromolecules, and mixtures thereof; andintroducing said activated phagocytic cells into said animal's body. 16.The method of claim 15 wherein said agent capable of causing said bodyto increase its activity of recognizing and removing macromolecules thathave undergone advanced glycosylation is in combination with aco-stimulatory agent which potentiates the activity of said agent. 17.The method of claim 16 wherein said advanced glycosylation endproductcomprises the reaction product of a proteinaceous macromolecule and asugar.
 18. The method of claim 16 wherein said advanced glycosylationendproduct is selected from the group consisting of the reaction productof albumin and glucose, the reaction product of albumin andglucose-6-phosphate, .Iadd.the fluorescent chromophore2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole bound to a carrier,.Iaddend.and mixtures thereof.
 19. The method of claim 16 wherein saidcarrier is selected from the group consisting of carbohydrates,proteins, lipids, synthetic polypeptides, biocompatible natural andsynthetic resins, antigens, and mixtures thereof.
 20. The method ofclaim 16 wherein said monokine comprises the polypeptide identified astumor necrosis factor.
 21. The method of claim 16 wherein saidco-stimulatory agent is selected from the group consisting ofInterleukin-1 and gamma-interferon.
 22. The method of claim 15 whereinsaid treated cells are introduced to said body by parenteral means. 23.The method of claim 22 wherein said parenteral means comprisesinjection.
 24. The method of claim 22 wherein said parenteral meanscomprises catheterization.
 25. The method of claim 15 including theparenteral administration of said agent to said body.
 26. The method ofclaim 15 wherein said body contains a quantity of insulin, and saidmethod includes the treatment of said body to lower the available activeinsulin level thereof.